ES2360858T3 - THIOPHEN DERIVATIVES AS CHK INHIBITORS 1. - Google Patents

Abstract

(S) 5- (3-Fluoro-phenyl) -3-ureido-thiophene-2-carboxylic acid, piperidin-3-ylamide, or a pharmaceutically acceptable salt thereof.

Description

Field of the Invention

The present invention relates to a novel substituted heterocycle, its pharmaceutical compositions and its use in the preparation of a medicament for the treatment and prevention of cancers.

Background of the invention

Chemotherapy and radiation exposure are currently the main options for cancer treatment, but the usefulness of these two approaches is seriously limited by the drastic adverse effects they have on normal tissues and the frequent development of resistance to tumor cells. Therefore, it is quite desirable to improve the effectiveness of such treatments in a manner that does not increase the toxicity associated with them. One way to achieve this is through the use of specific sensitizing agents such as those described herein.

An individual cell replicates by making an exact copy of its chromosomes and then segregating them into separate cells. This cycle of DNA replication, separation and division of chromosomes is regulated by mechanisms present in the cell that maintain the order of the stages and ensure that each stage is carried out precisely. The cell cycle control points are key in these processes (Hartwell et al., Science, Nov 3, 1989, 246 (4930): 629-34) at which cells can stand to ensure that DNA repair mechanisms have time to operate before continuing through the cycle towards mitosis. There are two control points like these in the cell cycle - the G 1 / S control point that is regulated by p53 and the G2 / M control point that is monitored by the kinase of control point 1 (CHK1) which is a Ser / Thr kinase.

Since cell cycle arrest induced by these control points is a crucial mechanism by which cells can overcome damage from radio- or chemo-therapy, their cancellation by new agents should increase the sensitivity of tumor cells with respect to the therapies that damage DNA. In addition, specific cancellation in tumors of the G1 / S control point by mutations in p53 in most tumors can be exploited to provide tumor selective agents. One method of designing chemosensitizers that nullify the G2 / M control point is to develop key G2 / M CHK1 regulatory kinase inhibitors and this method has been shown to work in several proof of concept studies. (Koniaras et al., Oncogene, 2001, 20: 7453; Luo et al., Neoplasia, 2001, 3: 411; Busby et al., Cancer Res., 2000, 60: 2108; Jackson et al., Cancer Res. , 2000, 60: 566).

Smithkline Beecham Corporation describes 2-ureidothiophene compounds in WO03029241 and 3-ureidothiophene compounds in WO03028731 as inhibitors of CHK1. The present invention provides new CHK1 inhibitors with improved properties.

Compendium of the invention

In accordance with the present invention, applicants have hereby discovered a new compound that is a potent inhibitor of CHK1 kinase and therefore possesses the ability to prevent cell cycle stopping at the G2 / M control point in response to DNA damage. This compound is accordingly useful for its antiproliferative activity (such as anti-cancer) and is therefore useful in methods of treating the human or animal body. The invention relates to pharmaceutical compositions containing said compound and its use in the manufacture of medicaments for use in the production of an anti-proliferative effect in warm-blooded animals such as humans.

The present invention includes pharmaceutically acceptable salts or prodrugs of said compound. In addition, according to the present invention, applicants provide pharmaceutical compositions and a method for the use of said compound in the treatment of cancer.

Such properties are expected to be of value in the treatment of pathologies associated with cell cycle arrest and cell proliferation such as cancers (solid tumors and leukemia), fibroproliferative and differential disorders, psoriasis, rheumatoid arthritis, Kaposi's sarcoma, hemangioma, acute and chronic kidney disease, atheroma, atherosclerosis, arterial restenosis, autoimmune diseases, acute and chronic inflammation, bone diseases and eye diseases with proliferation of retinal vessels.

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Detailed description of the invention

Accordingly, the present invention provides a compound of formula (I) or a pharmaceutically acceptable salt thereof:

image 1

In another embodiment the present invention provides a compound having the formula (I) wherein one or more of the atoms is a radioisotope of the same element.

In another embodiment, the present invention provides a compound of the formula (I) or a pharmaceutically acceptable salt thereof, for use in the treatment or prophylaxis of disorders associated with cancer.

In another embodiment the present invention provides a compound of formula (I) or a pharmaceutically acceptable salt thereof for use in the treatment or prophylaxis of a neoplastic disease such as cervical cancer, head and neck cancer, breast carcinoma, ovaries, lung (not small cells), pancreas, colon, prostate or other tissues, as well as leukemia and lymphomas, tumors of the peripheral and central nervous system, and other types of tumors such as melanoma, fibrosarcoma and osteosarcoma.

In another embodiment, the present invention provides a compound of formula (I) or a pharmaceutically acceptable salt thereof for use in the treatment or prophylaxis of proliferative diseases (including autoimmune, inflammatory, neurological and cardiovascular diseases).

These medical uses are as follows: A method for limiting cell proliferation in humans or animals comprising administering to said human or animal being a therapeutically effective amount of a compound of formula (I) or a salt thereof pharmaceutically acceptable.

These medical uses are as follows: A method for inhibiting CHK1 kinase comprising administering to a human or animal being a therapeutically effective amount of a compound of formula (I) or a pharmaceutically acceptable salt thereof.

A method of treating a human or animal suffering from cancer which comprises administering to said human or animal being a therapeutically effective amount of a compound of formula (I) or a pharmaceutically acceptable salt thereof.

A method of prophylactic treatment of cancer comprising administering to a human or animal being in need of such treatment a therapeutically effective amount of a compound of formula (I) or a pharmaceutically acceptable salt thereof.

A method of treating a human or animal being suffering from a neoplastic disease such as cervical cancer, head and neck cancer, breast carcinoma, ovaries, lung (not small cell), pancreas, colon, prostate or other tissues, as well as leukemia and lymphomas, tumors of the central and peripheral nervous system, and other types of tumors such as melanomasarcomas that include fibrosarcoma and osteosarcoma, malignant brain tumors, which comprises administering to said human or animal being a therapeutically effective amount of a compound of formula

(I) or one of its pharmaceutically acceptable salts.

A method of treating a human or animal being suffering from a proliferative disease such as autoimmune, inflammatory, neurological and cardiovascular diseases comprising administering to said human or animal being a therapeutically effective amount of a compound of formula (I) or one of its pharmaceutically acceptable salts.

A method of treating cancer by administration to a human or animal being a compound of formula (I) or a pharmaceutically acceptable salt thereof and an anti-tumor agent.

A method of treating cancer by administration to a human or animal being a compound of formula (I) or a pharmaceutically acceptable salt thereof and an agent that damages DNA.

A method for the treatment of infections associated with cancer comprising administering to a human or animal being in need of such treatment a therapeutically effective amount of a compound of formula (I) or a pharmaceutically acceptable salt thereof.

A method for the prophylactic treatment of infections associated with cancer comprising administering to a human or animal being in need of such treatment a therapeutically effective amount of a compound of formula (I) or a pharmaceutically acceptable salt thereof.

Another embodiment of the present invention provides a pharmaceutical composition comprising a compound of formula (I) or a pharmaceutically acceptable salt thereof together with at least one pharmaceutically acceptable excipient, diluent or carrier.

In another embodiment the present invention provides the use of a compound of formula (I) or a pharmaceutically acceptable salt thereof in the preparation of a medicament.

In another embodiment, the present invention provides the use of a compound of the formula (I) or a pharmaceutically acceptable salt thereof in the preparation of a medicament for the treatment or prophylaxis of cancer.

In another embodiment the present invention provides the compound of formula (I) or a pharmaceutically acceptable salt thereof in the preparation of a medicament for the treatment or prophylaxis of a neoplastic disease such as carcinoma of the breast, ovaries, lung, colon, prostate or other tissues, as well as leukemia and lymphomas, including CLL and CML, peripheral and central nervous system tumors, and other types of tumors such as melanoma, fibrosarcoma and osteosarcoma.

In yet another embodiment the present invention provides the use of a compound of formula (I) or a pharmaceutically acceptable salt thereof in the preparation of a medicament for the treatment or prophylaxis of proliferative diseases that include autoimmune, inflammatory, neurological and cardiovascular diseases.

In another embodiment, the present invention provides the use of a compound of the formula (I) or a pharmaceutically acceptable salt thereof in the preparation of a medicament for use in inhibiting the activity of CHK1 kinase.

In another embodiment, the present invention provides the use of a compound of the formula (I) or a pharmaceutically acceptable salt thereof in the preparation of a medicament for use in limiting cell proliferation.

Definitions

The definitions shown in this section are intended to clarify the terms used throughout this application. In this section, the definition applies to a compound of formula (I) unless otherwise indicated. The expression "herein" refers to the complete application.

As used herein, "pharmaceutically acceptable" is used herein to refer to the compounds, materials, compositions and / or dosage forms that, within the scope of the medical criteria, are suitable for use in contact with the tissues of humans and animals without causing toxicity, irritation, excessive allergic response or other problems or complications, in proportion to a reasonable benefit / risk ratio.

When used herein, "pharmaceutically acceptable salts" refers to derivatives of the described compounds in which the starting compound is modified by making salts with acids or bases thereof. Examples of pharmaceutically acceptable salts include, but are not limited to, salts with mineral or organic acids of basic moieties such as amines; alkaline or organic salts of acidic moieties such as carboxylic acids; and the like Pharmaceutically acceptable salts include conventional non-toxic salts or quaternary ammonium salts of the starting compound formed, for example, from non-toxic inorganic or organic acids. For example, said conventional non-toxic salts include those derived from inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, sulfamic acid, phosphoric acid, nitric acid and the like; and salts prepared from organic acids such as acetic, propionic, succinic, glycolic, stearic, lactic, maleic, tartaric, citric, ascorbic, palmitic, maleic, hydroxyleleic, phenylacetic, glutamic, benzoic, salicylic, sulphanilic, 2-acetoxybenzoic , fumaric, toluenesulfonic, methanesulfonic, ethanedisulfonic, oxalic, isethionic and the like.

The pharmaceutically acceptable salts of the present invention can be synthesized from the starting compound containing a basic or acidic moiety by conventional chemical methods. Generally, said salts can be prepared by reacting the free acid or base forms of these compounds with a stoichiometric amount of the appropriate base or acid in water or in an organic solvent or in a mixture of the two; In general, non-aqueous media such as ether, ethyl acetate, ethanol, isopropanol or acetonitrile are preferred. Lists of suitable salts are found in Remington's Pharmaceutical Sciences, 17th ed., Mack Publishing Company, Easton, Pa., 1985, p. 1418

Combinations

The anticancer treatment defined herein may be applied as a single therapy or may involve, in addition to the compound of the invention, surgery and / or conventional radiotherapy and / or chemotherapy. Such chemotherapy may include one or more of the following categories of antitumor agents:

(i)

 other antiproliferative / antineoplastic drugs and associations thereof, as used in medical oncology, such as alkylating or electrodeposition agents (for example, cis-platinum, oxaliplatin, carboplatin, cyclophosphamide, nitrogen mustard, melphalan, chlorambucil, busulfan, temozolamide and nitrosoureas); antimetabolites (for example, gemcitabine and fludarabine, as well as antifolates such as fluoropyrimidines such as 5fluorouracil and tegafur, raltitrexed, methotrexate, cytosine arabinoside and hydroxyurea); antitumor antibiotics (for example, anthracyclines such as adriamycin, bleomycin, doxorubicin, daunomycin, epirubicin, idarubicin, mitomycin-C, dactinomycin and mitramycin); antimitotic agents (for example vinca alkaloids such as vincristine, vinblastine, vindesine and vinorrelbine and taxoids such as taxol and taxotre); topoisomerase inhibitors (for example epipodophyllotoxins such as etoposide and teniposide, amsacrine, topotecan, irinotecan and camptothecin);

(ii)

 cytostatic agents such as antiestrogens (for example tamoxifen, toremifene, raloxifene, droloxifene and idoxifene), negative estrogen receptor regulators (for example fulvestrant), antiandrogens (for example bicalutamide, flutamide, nilutamide and cyproterone acetate), or LH antagonists LHRH agonists (for example goserelin, leuprorelin and buserelin), progestogens (for example megestrol acetate), aromatase inhibitors (for example as anastrozole, letrozole, vorazol and exemestane) and 5α-reductase inhibitors such as finasteride;

(iii) agents that inhibit the invasion of cancer cells (for example metalloproteinase inhibitors such as marimastat and inhibitors of the function of the plasminogen activator receptor urokinase);

(iv)

 inhibitors of growth factor function, for example such inhibitors include antibodies against growth factors, antibodies against growth factor receptors (for example the anti-erbb2 trastuzumab [Herceptin ™] antibody and the anti-erbb1 cetuximab antibody ( [C225]), farnesyl transferase inhibitors, tyrosine kinase inhibitors and serine / threonine kinase inhibitors, for example inhibitors of the epidermal growth factor family (eg EGFR family tyrosine kinase inhibitors such as N- (3 -chloro-4-fluorophenyl) -7-methoxy-6- (3-morpholinopropoxy) quinazolin-4-amine (gefitinib, AZD1839), N- (3-ethynylphenyl) -6,7-bis (2-methoxyethoxy) quinazolin-4- amine (erlotinib, OSI-774) and 6-acrylamido-N- (3-chloro-4-fluorophenyl) -7- (3morpholinopropoxy) quinazolin-4-amine (CI 1033)), for example inhibitors of the factor family of platelet-derived growth and for example inhibitors of the growth factor family of hepatocytes;

(v)

 antiangiogenic agents such as those that inhibit the effects of vascular endothelial growth factor (for example, the anti-vascular endothelial cell growth factor antibody bevacizumab [Avastin ™], compounds such as those described in International Patent Applications WO 97/22596, WO 97/30035, WO 97/32856 and WO 98/13354) and compounds that work by other mechanisms (eg linomide, inhibitors of the function of integrin αvβ3 and angiostatin);

(saw)

 vascular damage agents such as Combretastatin A4 and compounds described in International Patent Applications WO 99/02166, WO 00/40529, WO 00/41669, WO 01/92224, WO 02/04434 and WO 02/08213;

(vii) antisense therapies, for example those directed at the targets listed above, such as ISIS 2503, an anti-ras antisense;

(viii) gene therapy strategies, which include for example strategies to replace abnormal genes such as abnormal p53 or BRCA1 or abnormal BRCA2, GDEPT (gene targeted enzyme prodrug therapy) strategies, such as those using cytosine deaminase, thymidine kinase or a bacterial nitroreductase enzyme, and strategies to increase the patient's tolerance to chemotherapy or radiotherapy, such as multiple drug resistance gene therapy; Y

(ix) immunotherapy strategies, which include, for example, ex vivo and in vivo strategies to increase the immunogenicity of the patient's tumor cells, such as transfection with cytokines such as interleukin 2, interleukin 4, or granulocyte and macrophage colony stimulating factor , strategies to decrease the energy of T cells, strategies that employ transfected immune cells such as dendritic cells transfected with cytokines, strategies that use tumor cell lines transfected with cytokines, and strategies that use anti-idiotypic antibodies.

Said joint treatment can be achieved by means of simultaneous, sequential or independent dosing of the individual components of the treatment. Such combination products employ the compounds of this invention within the dosage range described above and the other pharmaceutically active agent within its authorized dosage range.

Formulations

The compounds of the present invention can be administered orally, parenterally, buccally, vaginally, rectally, inhalation, insufflation, sublingual, intramuscular, subcutaneous, topical, intranasal, intraperitoneal, intrathoracic, intravenous, epidural, intrathecal, intracerebroventricular and by injection into joints

The dosage will depend on the route of administration, the severity of the disease, the age and weight of the patient and other factors normally considered by the attending physician, when determining the individual regimen and dosage level as the most appropriate for a particular patient.

An effective amount of a compound of the present invention for use in an infection therapy is an amount sufficient to symptomatically relieve a warm-blooded animal, particularly a human being, the symptoms of an infection, to slow the progression of the infection or to reduce the risk of worsening in patients with symptoms of infection.

To prepare pharmaceutical compositions from the compounds of this invention, inert, pharmaceutically acceptable carriers can be either solid or liquid. Solid form preparations include powders, tablets, dispersible granules, capsules, wafers and suppositories.

A solid carrier can be one or more substances, which can also act as diluents, flavoring agents, solubilizers, lubricants, suspending agents, binders or tablet disintegrating agents; It can also be an encapsulating material.

In powders, the vehicle is a finely divided solid, which is mixed with the finely divided active component. In the tablets, the active ingredient is mixed with the vehicle that has the necessary binding properties, in suitable proportions and is compacted in the desired shape and size.

To prepare suppository compositions, a low melting wax such as a mixture of fatty acid glycerides and cocoa butter is melted and the active ingredient is dispersed therein, for example, by stirring. The molten homogeneous mixture is then poured into molds of convenient size and allowed to cool and solidify.

Suitable vehicles include magnesium carbonate, magnesium stearate, talc, lactose, sugar, pectin, dextrin, starch, tragacanth, methylcellulose, sodium carboxymethylcellulose, a low melting wax, cocoa butter and the like.

Some of the compounds of the present invention are capable of forming salts with various inorganic and organic acids and bases and such salts are also within the scope of this invention. Examples of such acid addition salts include acetate, adipate, ascorbate, benzoate, benzenesulfonate, bicarbonate, bisulfate, butyrate, canforate, canfosulfonate, choline, citrate, cyclohexyl sulfamate, diethylene diamine, ethanesulfonate, fumarate, glutamate, glycolate, hemisulfate, 2-hydroxyethyl sulphonate, heptanoate, hexanoate, hydrochloride, hydrobromide, hydroiodide, hydroxyleate, lactate, malate, maleate, methanesulfonate, meglumine, 2-naphthalenesulfonate, nitrate, oxalate, pamoate, persulfate, phenylacetate, phosphate, diphosphate, picrate, picrate, picrate quinate, salicylate, stearate, succinate, sulfamate, sulphanilate, sulfate, tartrate, tosylate (p-toluenesulfonate), trifluoroacetate and undecanoate. Base salts include ammonium salts, alkali metal salts such as sodium, lithium and potassium salts, alkaline earth metal salts such as aluminum, calcium and magnesium, salts with organic bases such as dicyclohexylamine salts, N-methyl- Dglucamine, and salts with amino acids such as arginine, lysine, ornithine, and so on. Likewise, the basic groups containing nitrogen can be quaternized with agents such as: lower alkyl halides, such as methyl, ethyl, propyl and butyl halides; dialkyl sulfates such as dimethyl, diethyl, dibutyl, diamyl sulfates; long chain halides such as decyl, lauryl, myristyl and stearyl halides; aralkyl halides such as benzyl bromide and others. Physiologically acceptable non-toxic salts are preferred, although other salts such as in isolation or purification of the product are also useful.

The salts may be formed by conventional means, such as by reacting the free base form of the product with one or more equivalents of the appropriate acid in a solvent or medium in which the salt is insoluble, or in a solvent such as water, which is removed under vacuum or by lyophilization or by exchanging the anions of an existing salt for another anion in a suitable ion exchange resin.

In order to use a compound of the formula (I) or a pharmaceutically acceptable salt thereof for therapeutic treatment (including prophylactic treatment) of mammals, including humans, it is usually formulated according to standard pharmaceutical practice as a composition. Pharmaceutical

In addition to the compounds of the present invention, the pharmaceutical composition of this invention may also contain, or co-administer (simultaneously or sequentially) with, one or more valuable pharmacological agents to treat one or more of the pathologies referred to herein.

The term "composition" is intended to include the formulation of the active component or a pharmaceutically acceptable salt with a pharmaceutically acceptable carrier. For example, this invention can be formulated by means known in the art in the form, for example, of tablets, capsules, aqueous or oily solutions, suspensions, emulsions, creams, ointments, gels, nasal sprays, suppositories, finely divided powders or aerosols. or nebulizers for inhalation and for parenteral use (including intravenous, intramuscular or infusion) sterile aqueous or oily solutions or suspensions or sterile emulsions.

Compositions in liquid form include solutions, suspensions and emulsions. Sterile solutions in water

or water-propylene glycol of the active compounds can be mentioned as an example of liquid preparations suitable for parenteral administration. Liquid compositions can also be formulated as a solution in aqueous polyethylene glycol solution. Aqueous solutions for oral administration can be prepared by dissolving the active component in water and adding suitable colorants, flavoring agents, stabilizers and thickening agents as desired. Aqueous suspensions for oral use can be prepared by dispersing the finely divided active ingredient in water together with a viscous material such as synthetic and natural gums, resins, methylcellulose, sodium carboxymethylcellulose and other suspending agents known in the art of pharmaceutical formulation.

The pharmaceutical compositions may be in unit dosage form. In such form, the composition is divided into unit doses containing appropriate amounts of the active component. The unit dose form may be a packaged preparation, the package containing discrete amounts of the preparations, for example, packaged tablets, capsules and powders in vials or ampoules. The unit dosage form may also be a capsule, wafer or tablet itself or it may be the appropriate number of any of these packaged forms.

The compound of formula (I) has been shown to inhibit the kinase activity of the control point in vitro. It has been shown that control point kinase inhibitors allow cells to progress inappropriately to the metaphase of mitosis resulting in apoptosis of the affected cells and, therefore, have antiproliferative effects. Therefore, it is believed that the compound of formula (I) and its pharmaceutically acceptable salts can be used for the treatment of neoplastic disease as described above. In addition, the compound of formula (I) and its pharmaceutically acceptable salts are also expected to be useful for the treatment of other proliferative diseases. It is expected that the compound of formula (I) will most likely be used in combination with a wide range of agents harmful to DNA but that could also be used as a single agent.

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Control Point Kinase 1 Assay: This in vitro assay measures the inhibition of CHK1 kinase by the compounds. The kinase domain is expressed in baculovirus and purified by the GST tag. The purified protein and biotinylated peptide substrate (Cdc25C) are then used in a Scintillation Proximity Test (SPA) in 384 automated wells. Specifically, the peptide, enzyme and reaction buffer are mixed and aliquoted in a 384 well plate containing dilution series of the compounds and controls. Hot and cold ATP are added to start the reaction. After 2 hours, a suspension of SPA, CsC12 and EDTA beds is added to stop the reaction and to capture the biotinylated peptide. The plates are counted in a Topcount. Data are analyzed and IC50s are determined for individual compounds.

Cancellation Assay: This cell assay measures the ability of CHK1 inhibitors to cancel the G2 / M control point induced by DNA damage. The compounds active against the enzyme (<2 uM) are tested in the cell assay. Briefly, HT29 cells (colon cancer cell line, without p53) are plated in 96-well plates on day 1. The next day, cells are treated with camptothecin for 2 hours to induce DNA damage. After 2 hours, the camptothecin is removed and the cells are treated for an additional 18 hours with the test compound and nocodazole, a toxic of the achromatic spindle that traps the cells in mitosis by canceling the control point. The cells are fixed with formaldehyde, stained for the presence of phosphohistone H3, a specific marker for mitosis, and marked with a Hoechst marker so that the number of cells can be measured. The plates are scanned using the Mitotic Index protocol in the Array Scan (Cellomics). As a positive control for cancellation, 4 mM caffeine is used. The compounds are tested in a response of 12 triplicate doses. Data are analyzed and EC50 are determined for individual compounds.

The invention will now be described in more detail with respect to the following illustrative example in which, unless otherwise indicated:

(i)

 temperatures are given in degrees Celsius (° C); operations are carried out at room or room temperature, that is, in a range of 18-25 ° C;

(ii)

 The organic solutions were dried over anhydrous sodium sulfate; evaporation of the organic solvent was carried out using a rotary evaporator under reduced pressure of 600-4000 Pa (4.5-30 mmHg) with a bath temperature of up to 60 ° C;

(iii) chromatography means flash chromatography on silica gel; Thin layer chromatography (TLC) was performed on silica gel plates;

(iv)

 in general, the progress of the reactions was followed by TLC or liquid chromatography / mass spectroscopy (LC-MS) and reaction times are given by way of illustration only,

(v)

 the final products have satisfactory proton nuclear magnetic resonance (NMR) spectra and / or mass spectral data;

(saw)

 yields are given only as an illustration and are not necessarily those that can be obtained through diligent process development; the preparations were repeated if more material was required;

(vii) when given, the NMR data are in the form of delta values for major diagnostic protons, given in parts per million (ppm) in relation to tetramethylsilane (TMS) as an internal standard, determined at 300 MHz, in DMSO- d6 unless otherwise indicated.

(viii) chemical symbols have their usual meanings;

(ix)

 Solvent ratios are given in terms of volume: volume (v / v);

(x)

 Rochelle salt is potassium and sodium tartarate;

(xi)

 Hunig's base is diisopropylethylamine (DIEA);

(xii) the purification of the compounds was carried out using one or more of the following procedures:

a) gas chromatography on normal silica gel; b) flash chromatography on silica gel using the Isco Combiflash® separation system: RediSep normal phase ultrafast column, flow rate, 30-40 ml / min;

c) flash chromatography on silica gel using the Biotage® separation system;

c) Gilson semiprep HPLC separation system: YMC ODS-AQ pack column, 100x20 mm, S 5µm 12 nm, water (0.1% trifluoroacetic acid) and acetonitrile (0.1% trifluoroacetic acid) as solvents, 20 min of operation; Y

(xvi) the following abbreviations have been used:

DMF EtOAc ether THF MeOH

dimethylformamide; ethyl acetate; diethyl ether; tetrahydrofuran; methanol

Example 1

(S) 5- (3-Fluoro-phenyl) -3-ureido-thiophene-2-carboxylic acid (piperidin-3-ylamide)

Methyl 3 - ({[(Trichloroacetyl) amino] carbonyl} amino) thiophene-2-carboxylate.

To a stirred solution of methyl 3-aminothiophene-2-carboxylate (17.0 g, 108 mmol) in anhydrous THF (216 mL) trichloroacetyl isocyanate (12.9 mL, 108 mmol) was added slowly over a period of 20 min. After finishing the addition, a precipitate formed and the reaction was stirred for an additional 1h at room temperature. The desired product was obtained by filtration to provide the title compound (99%) as a broken white solid. The product was used in the next step without further purification. LC / MS (ES, M + H = 345).

Methyl 5-Bromo-3 - ({[(trichloroacetyl) amino] carbonyl} amino) thiophene-2-carboxylate.

To a solution of methyl 3 - ({[(trichloroacetyl) amino] carbonyl} amino) thiophene-2-carboxylate (10.11 g, 29.38 mmol) in glacial acetic acid (146 mL) at 0 ° C is added slowly a solution of bromine (3 equiv) in glacial acetic acid (38mL). The resulting cloudy solution was at 70 ° C for 3 h after which the mixture was cooled to rt. The desired product (white solid, 12 g, 97%) was collected by filtration and the filtrate was washed with ether and dried in vacuo. LC / MS (ES, M + H = 424).

Methyl 3 - [(Aminocarbonyl) amino] -5-bromothiophene-2-carboxylate.

A solution of methyl 5-bromo-3 - ({[(trichloroacetyl) amino) carbonyl} amino) thiophene-2-carboxylate (12g; 28.3 mmol) in dry MeOH (60 mL) was purged with gaseous NH3 at 0 ° C until the solution became clear. The mixture was stirred for 20 min at rt and evaporation provided the desired product (7.8 g, 99%) as a white solid. 1H NMR (300 MHz, DMSO-d6) δ ppm 9.35 (s, 1H), 8.10 (S, 1H), 6.90 br s, 2H), 3.82 (s, 3 H); LC / MS (ES, M + H = 280).

(3S) -3-1 - [({3 - [(Aminocarbonyl) amino] -5-bromo-2-thienyl} carbonyl) amino] piperidine-1-carboxylic acid tert-butyl ester.

To a solution of methyl 3 - [(aminocarbonyl) amino] -5-bromothiophene-2-carboxylate (1.6 g, 5.7 mmol) in dry THF (30 mL) was added a solution of [Me3Al / (3S ) -3-aminopiperidin-1-tert-butyl carboxylate] in THF (18 mL) (which was performed by adding Me3Al (17.2 mL, 34.4 mmol) to a solution of (3S) -3- tert-butyl aminopiperidine-1-carboxylate (3.7 g, 17.2 mmol) in THF at -78 ° C and the resulting yellow solution was stirred at this temperature for 15 min) and the resulting dark yellow solution was heated to ta and stirred overnight at this temperature. The reaction mixture was cooled with ice and a saturated Rochelle salt solution was added slowly to stop the reaction. The mixture was partitioned between EtOAc and H2O, the aqueous layer was extracted with EtOAc (3x) and the combined organic extracts were washed with H2O, brine and dried (MgSO4). Evaporation provided a light orange solid. Purification by Gilson (5% H2O → 95% H2O-MeCN) provided the desired product (1.0 g, 40%) as a broken white solid. 1H NMR (300 MHz, DMSO-d6; 10.06 (s, 1H), 8.02 (s, 1H), 7.89 (d, 1H), 6.71 (brs, 2H), 4.04 ( m, 1H), 3.72 (m, 2H), 2.74 (m, 2H), 1.81 (m, 1H), 1.68 (m, 1H), 1.52 (m, 1H), 1.37 (s, 9H), 1.34 (m, 1H); LC / MS (ES, M + H = 448).

(3S) -3 - ({[3 - [(Aminocarbonyl) amino] -5- (3-fluorophenyl) -2-thienyl] carbonyl} amino) piperidine-1-carboxylic acid tert-butyl ester.

(Route A)

(3S) -3 - [({3 - [(aminocarbonyl) amino] -5-bromo-2-thienyl} carbonyl) amino] piperidine-1-carboxylic acid tert-butyl ester (1,000 mg, 2.24 mmol), acid 3-Fluorobenzeneboronic (470.2 mg, 3.36 mmol) and cesium carbonate (2,919.7 mg, 8.96 mmol) were dissolved in a mixture of water (3 mL) and 1,4-dioxane (20 mL) . The solution was degassed under vacuum and nitrogen. Palladium (0) tetrakis triphenylphosphine (259 mg, 0.22 mmol) was added to the solution and the colorless reaction mixture was heated at 75 ° C with stirring for 1 hour. The aqueous layer was separated and ethyl acetate (20 mL) was added and the resulting solution was dried over anhydrous MgSO4.

After evaporation of the solvent, the residual solid was subjected to flash chromatography on silica gel (40 g) with 2-5% methanol in methylene chloride for 30 min of gradient. The main fraction contained traces of triphenylphosphine oxide and was re-subjected to flash chromatography under the same conditions to provide a light yellow solid (916 mg, 88%). MS ES + 463 (M + 1); 1H NMR (CDCl3, 300 MHz) δ 10.35 (s, 1H), 8.30 (s, 1H), 7.43-7.28 (m, 3H), 7.05 (m, 1H), 5 , 91 (brs, 1H), 4.79 (brs, 2H), 4.05 (m, 1H), 3.53-3.55 (m, 2H), 3,363.29 (m, 2H), 1, 89-1.49 (m, 13H).

(Route B)

(2E, Z) -3-Chloro-3- (3-fluorophenyl) acrylonitrile.

POCl3 (13,049 mL, 140 mmol) was added to DMF (21.7 mL, 280 mmol) with stirring and cooling while maintaining the temperature below 40 ° C. After completion of the addition, 3-fluoroacetophenone (8,587 mL, 70 mmol) was added slowly. The reaction mixture was heated to 50 ° C and hydroxylamine chloride (19,457 g, 280 mmol) was added portionwise. After each addition the temperature was allowed to rise and the evolution of the gas ceased. When the addition finished the internal temperature of the reaction mixture reached 140 ° C. The mixture was allowed to stir and soon solidified. Water / ice was added slowly and the solid that separated was filtered off, washed with water on the filter (6 × 50 mL) and the solid was taken up with ethyl acetate (60 mL), the solution was dried over MgSO4 anhydrous and the solvent was evaporated to provide a brown oil (12 g). The oil was subjected to flash chromatography on silica gel (120 g) with 15-25% ethyl acetate in hexanes over a 20 min gradient. The first elution fraction was collected and evaporated to dryness. The residue was recrystallized from petroleum ether (150 mL) to provide a broken white solid (3.334 g, 26%). MS ES + 182 (M + 1); 1H NMR (CDCl3, 300 MHz) δ 7.44-7.17 (m, 1H), 7.24-7.17 (m, 1H), 6.04 (s, 1H).

Methyl 3-amino-5- (3-fluorophenyl) thiophene-2-carboxylate.

25% by weight sodium methoxide (3,429 mL, 15 mmol) was dissolved in methanol (6 mL) and methyl thioglycolate (1,341 mL, 15 mmol) was added with stirring. To the brown solution was added in small portions (2E, Z) -3-chloro-3- (3fluorophenyl) acrylonitrile (2,724 g, 15 mmol) and the resulting cream mass was heated at 76 ° C at reflux for 20 min. Ethyl acetate (60 mL) was added to the cold suspension and the organic phase was washed with water (2 × 50 mL) followed by a diluted solution of bleach (50 mL) and saturated aqueous ammonium chloride (50 mL). The organic phase was dried over MgSO4 and the solvent was evaporated to provide a brown solid. The solid was subjected to flash chromatography on silica gel (120 g) with 20% v / v ethyl acetate in hexane for 25 min. The first fraction was the desired product that was obtained after evaporation of the solvents as a broken white solid (2.282 g, 60.5%). MS ES-250 (M-1); 1H NMR (CDCl3, 300 MHz) δ 7.36-7.24 (m, 3H), 7.07-7.06 (m, 1H), 6.76 (s, 1H), 5.48 (brs, 2H), 3.84 (s, 3H).

Methyl 3 - [(aminocarbonyl) amino] -5- (3-fluorophenyl) thiophene-2-carboxylate.

Methyl 3-amino-5- (3-fluorophenyl) thiophene-2-carboxylate (2,517 g, 10 mmol) was dissolved in dry THF and cooled to -78 ° C. Trichloroacetylisocyanate (1,311 mL, 11 mmol) was added with stirring and the reaction was allowed to reach room temperature overnight. Methanol (10 mL) was added slowly and the solvents evaporated under reduced pressure. The white residue was suspended in methanol (200 mL) and 7M ammonium in methanol (3 mL, 21 mmol) was added. After stirring at room temperature for one hour the solvents were evaporated to dryness to provide a white powder. The white powder was suspended in hot methanol (10 mL) and allowed to cool to room temperature. The solid was filtered and dried in air to provide a cream-colored powder (2,861 g, 97%). MS ES + 295 (M + 1); 1H NMR (DMSO-d6, 300 MHz) δ 9.24 (s, 1H), 8.33 (s, 1H), 7.54-7.50 (m, 3H), 7.31-7.24 ( m, 1H), 6.86 (brs, 2H), 3.84 (s, 3H).

(3S) -3 - ({[3 - [(aminocarbonyl) amino] -5- (3-fluorophenyl) -2-thienyl] -carbonyl} amino) piperidine-1-carboxylic acid tert-butyl ester

Methyl 3 - [(aminocarbonyl) amino] -5- (3-fluorophenyl) thiophene-2-carboxylate (1,177 g, 4 mmol) was dissolved in THF (20 mL) and cooled to -78 ° C. A solution of (3S) -3-aminopiperidine-1-carboxylic acid tert-butyl (3,096 mL, 9.6 mmol) in THF (14 mL) was cooled to -78 ° C and 2M trimethylaluminum in hexane (5 mL) was added , 10 mmol). An aliquot of aluminate 5 (20 mL, 10 mmol) was added to the previously prepared ester solution in THF and the solution was heated at reflux for 4 hours. The cold reaction mixture was washed with a 20% aqueous solution of Rochelle salt. The organic layer was separated and the aqueous layer was extracted with ethyl acetate (2 x 20 ml). The combined organic phase was dried and the solvents evaporated under reduced pressure. The residue was dissolved in ethyl acetate and washed with aqueous 1M KHSO4 (2 x 20 mL) and brine (2 x 20 mL). After drying over MgSO4 and evaporating the solvent, the residue was subjected to

10 flash chromatography on silica gel (120g) with 50-70% ethyl acetate in hexane in a 45 min gradient. The last elution fraction was the desired product, (3S) -3 - ({[3 - [(aminocarbonyl) amino] -5- (3-fluorophenyl) -2-thienyl] carbonyl} amino) piperidine-1-carboxylate tertiary butyl, which was isolated as a vitreous solid (724 mg, 39%). MS ES + 463 (M + 1); 1H NMR (DMSO-d6, 300 MHz) δ 10.02 (s, 1H), 8.29 (s, 1H), 7.96 (brs, 1H), 7.55-7.42 (m, 3H) , 7.24 (t, 1H), 6.67 (brs, 1H), 3.75 (brs, 3H), 2.74-2.72 (m, 2H), 1.83-1.54 (m , 3H), 1.32 (s, 9 + 2H).

15 were dissolved in methanol (7ml), 5- (3-fluoro-phenyl) -3-ureido-thiophene-2-carboxylic acid (S) -piperidin-3-ylamide,

(3S) -3 - ({[3 - [(aminocarbonyl) amino] -5- (3-fluorophenyl) -2-thienyl] carbonyl} amino) piperidine-1-carboxylic acid tert-butyl ester (916 mg, 1.98 mmol) and 4M HCl in dioxane (10 mL, 40 mmol) was added, cooling to 0 ° C and the solution was stirred for 3 hours. The solvent was evaporated under reduced pressure and the residue was taken up with methanol (20 mL) and evaporated to dryness. This procedure was repeated 5 times to provide a white powder. The solid was dissolved in water (10 mL) and filtered and the

The solvent was evaporated by lyophilization for 48 hours. It was obtained as a light yellow solid (669 mg 84%). MS ES + 363 (M + 1) .1H NMR (DMSO-d6, 300 MHz) δ 9.95 (s, 1H), 9.23 (brs, 2H), 8.28 (s, 1H), 8.21 (d, 1H), 7.55-7.42 (m, 3H), 7.24 (m, 1H), 4.19 (m, 1H), 3.95 (brs, 6H, water), 3, 27-3.14 (m, 2H), 2.90-2.82 (m, 2H), 1.89-1.55 (m, 4H).

Claims (11)

one.

(S) 5- (3-Fluoro-phenyl) -3-ureido-thiophene-2-carboxylic acid, piperidin-3-ylamide, or a pharmaceutically acceptable salt thereof.

2.

The use of the compound of claim 1 or a pharmaceutically acceptable salt thereof and an antitumor agent in the preparation of a medicament for the treatment of cancer.

3.

The use of the compound of claim 1 or a pharmaceutically acceptable salt thereof and an agent that damages DNA in the preparation of a medicament for the treatment of cancer.

Four.

A pharmaceutical composition comprising the compound of claim 1 or a pharmaceutically acceptable salt thereof together with at least one pharmaceutically acceptable excipient, diluent or carrier.

5.

The use of the compound of claim 1 or a pharmaceutically acceptable salt thereof in the preparation of a medicament.

6.

The use of the compound of claim 1 or a pharmaceutically acceptable salt thereof in the preparation of a medicament for the treatment of cancer.

7.

The use of the compound of claim 1 or a pharmaceutically acceptable salt thereof in the preparation of a medicament for the treatment or prophylaxis of a neoplastic disease such as carcinoma of the breast, ovaries, lung, colon, prostate or other tissues, as well as leukemia. and lymphomas, including CLL and CML, peripheral and central nervous system tumors, and other types of tumors such as melanoma, fibrosarcoma and osteosarcoma.

8.

The use of the compound of claim 1 or a pharmaceutically acceptable salt thereof in the preparation of a medicament for the treatment or prophylaxis of proliferative diseases that include autoimmune, inflammatory, neurological and cardiovascular cardiovascular diseases.

9.

The use of the compound of claim 1 or a pharmaceutically acceptable salt thereof in the preparation of a medicament for use in inhibiting the activity of CHK1 kinase.

10.

The use of the compound of claim 1 or a pharmaceutically acceptable salt thereof in the preparation of a medicament for use in limiting cell proliferation.

eleven.

A compound according to claim 1 or a pharmaceutically acceptable salt thereof for use in the treatment or prophylaxis of disorders associated with cancer.